The transcription factor cAMP response element-binding protein (CREB1) has been shown to be involved in diverse biological pathways including the regulation of cell proliferation, apoptosis, cell ...cycle progression, and metastasis. In this context, aberrant expression of CREB1 and the functional consequences are well investigated in a number of hematopoietic and solid tumors. However, CREB1 expression and underlying control mechanisms are only poorly analyzed in renal cell carcinoma (RCC). The present study confirmed a deregulation of CREB1 protein in the clear cell type of RCC (ccRCC) and analysis of in-house ccRCC cell lines suggested a post-transcriptional control. The combination of miRNA enrichment assay, in silico analysis and molecular biological approaches revealed four novel CREB1-regulating miRNAs, namely miR-22-3p, miR-26a-5p, miR-27a-3p, and miR-221-3p. Categorizing RCC samples as CREB1 negative or positive, respectively, the expression of these miRNAs was found to be inversely correlated with CREB1 protein levels. Analyzing 453 consecutive RCC tumors by immunohistochemistry, weakly negative, but significant correlations of CREB1 with tumor stage and grade, vascular invasion (V1) and lymphovascular invasion (L1) were found. In this respect, ccRCC might differ from other solid tumors like esophageal squamous-cell carcinoma or glioma.
Purpose
Recently discovered molecular classifications for urothelial bladder cancer appeared to be promising prognostic and predictive biomarkers. The present study was conducted to evaluate the ...prognostic impact of molecular subtypes assessed by two different methodologies (gene and protein expression), to compare these two approaches and to correlate molecular with histological subtypes in a consecutively collected, mono-institutional muscle-invasive bladder cancer (MIBC) cohort.
Methods
193 MIBC were pathologically re-evaluated and molecular subtypes were assessed on mRNA (NanoString technology, modified 21-gene-containing MDACC approach) and protein levels (immuno-histochemical IHC analysis of CK5, CK14, CD44, CK20, GATA3 and FOXA1). Descriptive statistical methods and uni-/multi-variable survival models were employed to analyze derived data.
Results
Neither gene expression nor protein-based subtyping showed significant associations with disease-specific (DSS) or recurrence-free survival (RFS). Agreement between mRNA (reference) and protein-based subtyping amounted 68.6% for basal, 76.1% for luminal and 50.0% for double-negative tumors. Histological subtypes associated with RFS in uni-variable (
P
= 0.03), but not in multivariable survival analyses. Tumors with variant histology predominantly showed luminal subtypes (gene expression subtyping: 36/55 cases, 65.5%; protein subtyping: 44/55 cases, 80.0%). Squamous differentiation significantly associated with basal subtypes (gene expression subtyping: 44/45 squamous cases, 97.8%; protein subtyping: 36/45 cases, 80.0%).
Conclusion
In our consecutive cystectomy cohort, neither gene, protein expression-based subtyping, nor histological subtypes associated with DSS or RFS in multi-variably adjusted survival analyses. Application of a limited IHC subtyping marker panel showed high concordance of 83.9% with gene expression-based subtyping, thus underlining the utility for subtyping in pathological routine diagnostics. In addition, histological MIBC subtypes are strong indicators for intrinsic subtypes.
The Piwi-like genes belong to the Argonaute gene family and are conserved in plants, animals and humans. In addition to their essential role in the germ line and as stem cell-associated genes, ...Piwi-like proteins play a role in different cancer types but have yet to be studied in renal cell carcinoma (RCC). We investigated tissue micro arrays (TMAs) with tumor samples of two independent cohorts of RCC patients (N = 265 and N = 345); we used immunohistochemistry to assess the protein expression of Piwi-like 1. Applying an immunoreactive score (IRS), we found Piwi-like 1 positivity (IRS > 0) in 28.3% and 14.8% of the tumors in cohorts 1 and 2, respectively. Piwi-like 1 positivity was correlated with Fuhrman grade, tumor stage and the presence of distant metastasis (P < 0.005). Moreover, in univariate and multivariate analyses (adjusted to Fuhrman grade and tumor stage), Piwi-like 1 positivity was associated with a shorter cancer-specific survival in the patients in the second cohort. In addition, Piwi-like 1 expression allowed to further distinguish the RCC patients with high Fuhrman grade, high tumor stage, distant metastasis or high pre-operative levels of C-reactive protein, as Piwi-like 1 positivity was associated with a shorter cancer-specific survival in both cohorts. Our data encourage further investigations to enlighten the role of Piwi-like 1 and its function as a marker of poor prognosis in RCC patients.
The androgen/androgen receptor (AR)-signaling axis plays a central role in prostate cancer (PCa). Upon androgen-binding the AR dimerizes with another AR, and translocates into the nucleus where the ...AR-dimer activates/inactivates androgen-dependent genes. Consequently, treatments for PCa are commonly based on androgen deprivation therapy (ADT). The clinical benefits of ADT are only transitory and most tumors develop mechanisms allowing the AR to bypass its need for physiological levels of circulating androgens. Clinical failure of ADT is often characterized by the synthesis of a constitutively active AR splice variant, termed AR-V7. AR-V7 mRNA expression is considered as a resistance mechanism following ADT. AR-V7 no longer needs androgenic stimuli for nuclear entry and/or dimerization.
Our goal was to mechanistically decipher the interaction between full-length AR (AR-FL) and AR-V7 in AR-null HEK-293 cells using the NanoLuc Binary Technology under androgen stimulation and deprivation conditions.
Our data point toward a hypothesis that AR-FL/AR-FL homodimers form in the cytoplasm, whereas AR-V7/AR-V7 homodimers localize in the nucleus. However, after androgen stimulation, all the AR-FL/AR-FL, AR-FL/AR-V7 and AR-V7/AR-V7 dimers were localized in the nucleus.
We showed that AR-FL and AR-V7 form heterodimers that localize to the nucleus, whereas AR-V7/AR-V7 dimers were found to localize in the absence of androgens in the nucleus.
Bladder cancer (BC) is the 12th most commonly diagnosed cancer worldwide. Although there are several well-established molecular and immunological classifications, prognostic and predictive markers ...for tumor cells and immune cells are still needed. Using a tissue microarray, we analyzed the expression of the chemokine CC motif ligand 5 (CCL5) by immunohistochemistry (IHC) in 175 muscle-invasive BC samples. The application of a single cutoff for the staining status of tumor cells (TCs; positive vs. negative) and immune cells (ICs; positive vs. negative) revealed 75 patients (42.9%) and 123 patients (70.3%) with CCL5-positive TCs or ICs, respectively. IHC results were associated with prognostic and predictive data. Multivariate Cox regression analysis revealed that positive CCL5 staining in TCs was associated with significantly shorter disease-specific survival (DSS; RR = 1.51; p = 0.047), but CCL5-negative ICs were associated with significantly shorter overall survival (OS; RR = 1.66; p = 0.005), DSS (RR = 2.02; p = 0.001) and recurrence-free survival (RFS; RR = 1.94; p = 0.002). Adjuvant chemotherapy was favorable for patients with CCL5-negative ICs for OS (RR = 0.30; p = 0.006), DSS (RR = 0.36; p = 0.022) and RFS (RR = 0.41; p = 0.046) but not for patients with CCL5-positive ICs, except in the subgroup of N1 + N2 patients, where it was associated with better OS. We suggest that CCL5 expression can be a prognostic and predictive marker for muscle-invasive bladder cancer patients.
The non-classical human leukocyte antigen G (HLA-G) is a potent regulatory protein involved in the induction of immunological tolerance. This is based on the binding of membrane-bound as well as ...soluble HLA-G to inhibitory receptors expressed on various immune effector cells, in particular NK cells and T cells, leading to their attenuated functions. Despite its restricted expression on immune-privileged tissues under physiological conditions, HLA-G expression has been frequently detected in solid and hematopoietic malignancies including urological cancers, such as renal cell and urothelial bladder carcinoma and has been associated with progression of urological cancers and poor outcome of patients: HLA-G expression protects tumor cells from anti-tumor immunity upon interaction with its inhibitory receptors by modulating both the phenotype and function of immune cells leading to immune evasion. This review will discuss the expression, regulation, functional and clinical relevance of HLA-G expression in urological tumors as well as its use as a putative biomarker and/or potential therapeutic target for the treatment of renal cell carcinoma as well as urothelial bladder cancer.
The tumor suppressor p53 gene is mutated in minimally half of all cancers. It is therefore reasonable to assume that naturally occurring polymorphic genetic variants in the p53 stress response ...pathway might determine an individual's susceptibility to cancer. A central node in the p53 pathway is the MDM2 protein, a direct negative regulator of p53. In this report, a single nucleotide polymorphism (SNP309) is found in the MDM2 promoter and is shown to increase the affinity of the transcriptional activator Sp1, resulting in higher levels of MDM2 RNA and protein and the subsequent attenuation of the p53 pathway. In humans, SNP309 is shown to associate with accelerated tumor formation in both hereditary and sporadic cancers. A model is proposed whereby SNP309 serves as a rate-limiting event in carcinogenesis.
The co-expression of miRNAs and their target proteins was studied on tissue microarrays from different prostate cancer (PCa) patients. PCa of primary Gleason pattern 4 (GP4), lymph node metastases of ...GP4, distant metastases, and normal tissue from the transitional and peripheral zones were co-stained by fluorescent miRNA in situ hybridization (miRisH) and protein immunohistofluorescence (IHF). The miRNAs and corresponding target proteins include the pairs miR-145/ERG, miR-143/uPAR, and miR-375/SEC23A. The fluorescence-stained and scanned tissue microarrays (TMAs) were evaluated by experienced uropathologists. The pair miR-145/ERG showed an exclusive staining for miR-145 in the nuclei of stromal cells, both in tumor and normal tissue, and for ERG in the cytoplasm with/without co-expression in the nucleus of tumor cells. The pair miR-143/uPAR revealed a clear distinction between miR-143 in the nuclei of stromal cells and uPAR staining in the cytoplasm of tumor cells. Metastases (lymph node and distant) however, showed tumor cells with cytoplasmic staining for miR-143/uPAR. In normal tissues, beside the nuclei of the stroma cells, gland cells could also express miR-143 and uPAR in the cytoplasm. miR-375 showed particular staining in the nucleoli of GP4 and metastatic samples, suggesting that nucleoli play a special role in sequestering proteins and miRNAs. Combined miRisH/IHF allows for the study of miRNA expression patterns and their target proteins at the single-cell level.
Genome-wide analyses revealed basal and luminal subtypes of urothelial carcinomas of the bladder. It is unknown if this subtyping can also be applied to upper tract urothelial carcinomas.
Tumor ...samples from 222 patients with upper tract urothelial carcinomas who were treated with radical nephroureterectomy were analyzed for the expression of seven basal/luminal immunohistochemical markers (CK5, EGFR, CD44, CK20, p63, GATA3, FOXA1).
Hierarchical clustering revealed a basal-like subtype (enrichment of CK5, EGFR and CD44) in 23.9% and a luminal-like subtype (enrichment of CK20, GATA3, p63 and FOXA1) in 13.1% of the patients. In 60.8%, little to no markers were expressed, whereas markers of both subtypes were expressed in 2.2%. By using CK5 and CK20 as surrogate markers for the basal and luminal subtypes, we defined four subtypes of upper tract urothelial carcinomas: (i) exclusively CK20 positive and CK5 negative (CK20+/CK5-), (ii) exclusively CK5 positive and CK20 negative (CK20-/ CK5+), (iii) both markers positive (CK20+/CK5+) and (iv) both markers negative (CK20-/CK5-). A receiver-operator analysis provided the optimal cut-off values for this discrimination. An immunoreactive score >1 for CK5 and >6 for CK20 were defined as positive. In multivariate Cox's regression analysis, the CK20+/CK5- subtype was an independent negative prognostic marker with a 3.83-fold increased risk of cancer-specific death (p = 0.02) compared to the other three subtypes.
Immunohistochemical subgrouping of upper tract urothelial carcinomas by analyzing CK5 and CK20 expression can be performed in a routine setting and can identify tumors with a significantly worse cancer-specific survival prognosis.
s
Carbonic anhydrase 9 (CAIX) is an important protein that stabilizes the extracellular pH value and is transcriptionally regulated by hypoxia-inducible factor 1 (HIF1), but more stable than HIF1α. ...Here we show a comparative study that examines the prognostic value of CA9 mRNA, CAIX protein of tumor cells and secreted CAIX protein for oral squamous cell carcinoma (OSCC) patients. Tumor samples from 72 OSCC patients and 24 samples of normal tissue were analyzed for CA9 mRNA levels. A total of 158 OSCC samples were stained for CAIX by immunohistochemistry and 89 blood serum samples were analyzed by ELISA for soluble CAIX protein content. Survival analyses were performed by Kaplan⁻Meier and Cox's regression analysis to estimate the prognostic effect of CA9/CAIX in OSCC patients. The CA9 mRNA and CAIX protein levels of tumor cells correlated with each other, but not with those of the secreted CAIX protein level of the blood of patients. ROC curves showed a significant (
< 0.001) higher mRNA-level of CA9 in OSCC samples than in adjacent normal tissue. Cox's regression analysis revealed an increased risk (i) of death for patients with a high CA9 mRNA level (RR = 2.2; p = 0.02), (ii) of locoregional recurrence (RR = 3.2; p = 0.036) at higher CA9 mRNA levels and (iii) of death at high CAIX protein level in their tumors (RR = 1.7; p = 0.066) and especially for patients with advanced T4-tumors (RR = 2.0; p = 0.04). However, the secreted CAIX protein level was only as a trend associated with prognosis in OSCC (RR = 2.2; p = 0.066). CA9/CAIX is an independent prognostic factor for OSCC patients and therefore a potential therapeutic target.
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